Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

Activity-Based Protein Profiling of Non-ribosomal Peptide Synthetases.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Chemical Profiling of the Endoplasmic Reticulum Proteome Using Designer Labeling Reagents.

The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.

Purine Biosynthesis Enzymes in Hippocampal Neurons.

Despite reports implicating disrupted purine metabolism in causing a wide spectrum of neurological defects, the mechanistic details of purine biosynthesis in neurons are largely unknown. As an initial step in filling that gap, we examined the expression and subcellular distribution of three purine biosynthesis enzymes (PFAS, PAICS and ATIC) in rat hippocampal neurons. Using immunoblotting and high-resolution light and electron microscopic analysis, we find that all three enzymes are broadly distributed in hippocampal neurons with pools of these enzymes associated with mitochondria. These findings suggest a potential link between purine metabolism and mitochondrial function in neurons and provide an impetus for further studies.

A Chemoproteomics Approach to Investigate Phosphopantetheine Transferase Activity at the Cellular Level.

Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4'-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.

Small Molecules from Deep within the Gut.

Although the influence of the human microbiome on host functions is widely recognized, the underlying molecular mechanisms are still largely unknown. A recent study by the Fischbach group now provides an experimental workflow for characterizing and evaluating the impact of microbiome-derived small molecules on host physiology.

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products.

Functional profiling of adenylation domains in nonribosomal peptide synthetases by competitive activity-based protein profiling.

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.

Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols.

The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.

Molecular docking and molecular dynamics studies on ß-lactamases and penicillin binding proteins.

Bacterial resistance to ß-lactam antibiotics poses a serious threat to human health. Penicillin binding proteins (PBPs) and ß-lactamases are involved in both antibacterial activity and mediation of ß-lactam antibiotic resistance. The two major reasons for resistance to ß-lactams include: (i) pathogenic bacteria expressing drug insensitive PBPs rendering ß-lactam antibiotics ineffective and (ii) production of ß-lactamases along with alteration of their specificities. Thus, there is an urgent need to develop newer ß-lactams to overcome the challenge of bacterial resistance. Therefore the present study aims to identify the binding affinity of ß-lactam antibiotics with different types of PBPs and ß-lactamases. In this study, cephalosporins and carbapenems are docked into PBP2a of Staphylococcus aureus, PBP2b and PBP2x of Streptococcus pneumoniae and SHV-1 ß-lactamase of Escherichia coli. The results reveal that Ceftobiprole can efficiently bind to PBP2a, PBP2b and PBP2x and not strongly to SHV-1 ß-lactamase. Furthermore, molecular dynamics (MD) simulations are performed to refine the binding mode of the docked complex structure and to observe the differences in the stability of free PBP2x and Ceftobiprole bound PBP2x. MD simulation supports the greater stability of the Ceftobiprole-PBP2x complex compared to free PBP2x. This work demonstrates that potential ß-lactam antibiotics can efficiently bind to different types of PBPs for circumventing ß-lactam resistance and opens avenues for the development of newer antibiotics that can target bacterial pathogens.

Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody.

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.

Evolutionary imprint of catalytic domains in fungal PKS-NRPS hybrids.

Fungal hybrid enzymes consisting of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) module are involved in the biosynthesis of a vast array of ecologically and medicinally relevant natural products. Whereas a dozen gene clusters could be assigned to the requisite PKS-NRPS pathways, the programming of the multifunctional enzymes is still enigmatic. Through engineering and heterologously expressing a chimera of PKS (lovastatin synthase, LovB) and NRPS (cytochalasin synthase, CheA) in Aspergillus terreus, we noted the potential incompatibility of a fungal highly reducing PKS (hrPKS) with the NRPS component of fungal PKS-NRPS hybrids. To rationalize the unexpected outcome of the gene fusion experiments, we conducted extensive bioinformatic analyses of fungal PKS-NRPS hybrids and LovB-type PKS. From motif studies and the function of the engineered chimeras, a noncanonical function of C-terminal condensation (C) domains in truncated PKS-NRPS homologues was inferred. More importantly, sequence alignments and phylogenetic trees revealed an evolutionary imprint of the PKS-NRPS domains, which reflect the evolutionary history of the entire megasynthase. Furthermore, a detailed investigation of C and adenylation (A) domains provides support for a scenario in which not only the A domain but also the C domain participates in amino acid selection. These findings shed new light on the complex code of this emerging class of multifunctional enzymes and will greatly facilitate future combinatorial biosynthesis and pathway engineering approaches towards natural product analogues.

[Evaluation of the immunochromatography kit for detection of emetic-toxin producing Bacillus cereus].

We evaluated the sensitivity and specificity of an immunochromatography kit, Single-path Emetic Tox Mrk (Merck), which targets a marker protein for the detection of Bacillus cereus that produces emetic toxin. Strains were isolated after outbreaks of food poisoning, and from retail prepared foods and food products. The strains were examined for the presence of the emetic toxin-synthetase gene by PCR. All 58 emetic strains isolated from the food poisoning cases showed a positive reaction in the immunochromatography kit. No emetic strains gave false negative result. Among 47 non-emetic strains, only two strains isolated from the food poisonings and one strain isolated from food products showed a false positive reaction in the test. We concluded that this method has high sensitivity and specificity. The test can be used for detection of emetic toxin-producing B. cereus not only from food poisoning cases, but also in food products.

Isolation and 2-D-DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes.

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP-1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2-D-DIGE approach followed by MALDI-TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D-Ala-D-Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde-3-phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti-Listeria therapy.

An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.

Positional isotope exchange analysis of the Mycobacterium smegmatis cysteine ligase (MshC).

MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins, which is an intermediate in the biosynthetic pathway of mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH or AcCys-GlcN-Ins). MSH is produced by Mycobacterium tuberculosis, members of the Actinomycetes family, to maintain an intracellular reducing environment and protect against oxidative and antibiotic induced stress. The biosynthesis of MSH is essential for cell growth, and therefore, the MSH biosynthetic enzymes present potential targets for inhibitor design. The formation of kinetically competent adenylated intermediates was suggested by the observation of positional isotope exchange (PIX) reaction using [betagamma-(18)O6]-ATP in the presence of cysteine. The PIX rate depends on the presence of cysteine and increases with concentrations of cysteine. The loss of PIX activity upon the addition of small concentrations of pyrophosphatase suggests that the PP(i) is free to dissociate from the active site of cysteine ligase into the bulk solution. The PIX activity is also eliminated at high concentrations of GlcN-Ins, consistent with the mechanism in which GlcN-Ins binds after cysteine-adenylate formation. This PIX analysis confirms that MshC catalyzes the formation of a kinetically competent cysteinyl-adenylate intermediate after the addition of ATP and cysteine.

High-throughput profiling of posttranslational modification enzymes by phage display.

Phage display has been used as a high-throughput platform for identifying proteins or peptides with desired binding or catalytic activities from a complex proteome. Recently, phage display has been applied to profile the catalytic activities of posttranslational modification (PTM) enzymes. Here, we highlight recent work elucidating the downstream targets of PTM enzymes by phage display, including the genome-wide profiling of biosynthetic enzymes subject to phosphopantetheinyl transferase (PPTase) modification.

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA.

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.